Polyunsaturated liposomes are antiviral against hepatitis B and C viruses and HIV by decreasing cholesterol levels in infected cells.

The pressing need for broad-spectrum antivirals could be met by targeting host rather than viral processes. Cholesterol biosynthesis within the infected cell is one promising target for a large number of viral systems, including hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV. Liposomes developed for intracellular, endoplasmic reticulum (ER)-targeted in vivo drug delivery have been modified to include polyunsaturated fatty acids that exert an independent antiviral activity through the reduction of cellular cholesterol. These polyunsaturated ER liposomes (PERLs) have greater activity than lovastatin (Mevacor, Altoprev), which is clinically approved for lowering cholesterol and preventing cardiovascular disease. Treatment of HCV, HBV, and HIV infections with PERLs significantly decreased viral secretion and infectivity, and pretreatment of naïve cells reduced the ability of both HCV and HIV to establish infections because of the decreased levels of plasma membrane cholesterol. Direct competition for cellular receptors was an added effect of PERLs against HCV infections. The greatest antiviral activity in all three systems was the inhibition of viral infectivity through the reduction of virus-associated cholesterol. Our study demonstrates that PERLs are a broadly effective antiviral therapy and should be developed further in combination with encapsulated drug mixtures for enhanced in vivo efficacy.

All in the Family: Living With ADNP Syndrome.

PURPOSE: The overarching purpose of this research was to examine the experiences of 1 family living with a child with Helsmoortel-Van Der Aa syndrome or activity-dependent neuroprotective protein (ADNP) syndrome. DESIGN: A retrospective qualitative design was used for this study. METHODS: Two primary caregivers for a 5-year-old child with ADNP syndrome completed background questionnaires to provide context for semistructured interviews. Each caregiver completed 2 interviews, approximately 2 months apart. Field notes, member checks, and triangulation were used to enhance the credibility of the study. RESULTS: This article summarizes the theme "All in the Family." Having a child with ADNP syndrome affected all aspects of family life. Participants revealed that family dynamics were shaped by experiences stemming from their living arrangements and caregiving responsibilities. CONCLUSIONS: Findings from this research highlighted the need for increased support for families faced with ADNP syndrome, as well as the role clinical nurse specialists can play in the lives of caregivers faced with such a rare diagnosis. Furthermore, given the paucity of ADNP syndrome information, the need for more research is warranted.

Uptake and trafficking of liposomes to the endoplasmic reticulum.

Liposomes are vesicular structures consisting of an aqueous core surrounded by a lipid bilayer. Apart from the cytosol and lysosomes, no other intracellular compartment has been successfully targeted using liposomal delivery. Here, we report the development of liposomes capable of specific targeting to the endoplasmic reticulum (ER) and associated membranes. Using competition and inhibitor assays along with confocal microscopy, we have determined that ER liposomes utilize scavenger and low-density lipoprotein receptors for endocytosis and enter cells through a caveolin- and microtubule-dependent mechanism. They traffic intact to the ER, where fusion with the ER membrane occurs after 22-25 min, which was confirmed by fluorescence-dequenching assays. Once inside the ER, tagged lipids intercalate with the ER membrane and are subsequently incorporated into ER-assembling entities, such as the ER-budding viruses hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), lipid droplets, and secreted lipoproteins. ER liposomes are superior to cytosolic liposome formulations for the intracellular delivery of aqueous cargo, such as HIV-1 antivirals, and are especially suited for the prolonged delivery of lipids and lipophilic drugs into human cells.

Crystal structure of the bb' domains of the protein disulfide isomerase ERp57.

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.

PCR-based unidirectional deletion method for creation of comprehensive cDNA libraries.

A new strategy for the rapid creation of DNA deletion libraries using a simple PCR-based method is presented. Unidirectional deletion fragments are created and may be cloned into any vector system without the constraint of using restriction enzymes. Our strategy combines methodologies from DNA sequencing, PCR, and homologous recombination (either in vivo or in vitro) to allow for the creation of a library containing fragments representing all possible deletions of a given cDNA. Using this strategy we have successfully constructed a deletion library of the cDNA encoding for the lumenal domain of yeast Ire1p, and have shown that resulting fragments range from 100 bp to the full length cDNA (1557 bp). This method is simple, inexpensive, and can easily be adapted for automated high-throughput research.

DePIE: Designing Primers for Protein Interaction Experiments.

Several primer prediction and analysis programs have been developed for diverse applications. However, none of these existing programs can be directly used for the design of primers in protein interaction experiments, since proteins may have transmembrane domains (TMDs) and/or a signal peptide that must be excluded from experiments. Furthermore, it is frequently the case that a short restriction sequences must be added to each primer in order to clone PCR products into a given destination vectors for expression. DePIE, a web-based primer design tool, was developed to address these deficiencies. The program takes as input NCBI protein accession numbers and returns primer information including nucleotide sequences, thermodynamic melting temperature of the nucleotide sequences and the target positions. DePIE is implemented in JAVA, PERL and PHP and has proven to be very efficient in designing primers for our interaction experiments. DePIE services can be accessed at the web site: http://biocore.unl.edu/primer/primerPI.html.

N-Butyldeoxynojirimycin is a broadly effective anti-HIV therapy significantly enhanced by targeted liposome delivery.

OBJECTIVE: N-Butyldeoxynojirimycin (NB-DNJ), an inhibitor of HIV gp120 folding, was assessed as a broadly active therapy for the treatment of HIV/AIDS. Furthermore, to reduce the effective dose necessary for antiviral activity, NB-DNJ was encapsulated inside liposomes and targeted to HIV-infected cells. METHODS: Thirty-one primary isolates of HIV (including drug-resistant isolates) were cultured in peripheral blood mononuclear cells to quantify the effect of NB-DNJ on viral infectivity. pH-sensitive liposomes capable of mediating the intracellular delivery of NB-DNJ inside peripheral blood mononuclear cells were used to increase drug efficacy. RESULTS: NB-DNJ decreased viral infectivity with a single round of treatment by an average of 80% in HIV-1-infected and 95% in HIV-2-infected cultures. Two rounds of treatment reduced viral infectivity to below detectable levels for all isolates tested, with a calculated IC50 of 282 and 211 micromol/l for HIV-1 and HIV-2, respectively. When encapsulated inside liposomes, NB-DNJ inhibited HIV-1 with final concentrations in the nmol/l range (IC50 = 4 nmol/l), a 100 000-fold enhancement in IC50 relative to free NB-DNJ. Targeting liposomes to the gp120/gp41 complex with a CD4 molecule conjugated to the outer bilayer increased drug/liposome uptake five-fold in HIV-infected cells compared with uninfected cells. NB-DNJ CD4 liposomes demonstrated additional antiviral effects, reducing viral secretion by 81% and effectively neutralizing free viral particles to prevent further infections. CONCLUSION: The use of targeted liposomes encapsulating NB-DNJ provides an attractive therapeutic option against all clades of HIV, including drug-resistant isolates, in an attempt to prevent disease progression to AIDS.

Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system.

Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P-domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin-like repeats and a short basic C-terminal tail. Here we show direct interactions between the tip of the calnexin P-domain and the ERp57 basic C-terminus by using NMR and a novel membrane yeast two-hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P-domain is active in binding ERp57, and we determine the structure of the bound conformation of the P-domain peptide. The experimental strategy of using the MYTHS two-hybrid system to map interaction sites between ER proteins, together with NMR, provides a powerful new strategy for establishing the function of ER complexes.